Investigation of the genetics and epidemiology of idiopathic Parkinson's disease (IPD) have not identified an etiology. Risk of IPD is consistently higher among first-degree relatives of affected cases than among controls (1), but only a small number of families with multiple affected members have been reported suggesting an autosomal-dominant pattern with reduced penetrance (2). The concordance rate for IPD is higher for monozygotic than for dizygotic twin pairs in which one twin had onset before age 60 but not for pairs with a later age at onset (3). A cytochrome P450 gene, CYP2D6, was implicated as a candidate gene for IPD by some (5), but not all investigators (6), because the frequency of certain mutant alleles differed in IPD and controls (7,8). While each of these observations is compatible with a genetic etiology for IPD, the genetic contributions appear to be complex. The observations that occupational manganese exposure causes a form of parkinsonism (9,10) and that N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a form of parkinsonism in drug users (11) provide support for an environmental cause of IPD. Prevalence of IPD appears to be increased among individuals exposed to pesticides and related chemicals, especially MPTP-like compounds (12,13). IPD is also more prevalent among individuals raised in rural environments (14) or exposed to well water (15). Cigarette smoking (16) and the use of anti-oxidant vitamin supplements (17) remain unconfirmed "protective" factors. Thus, environmental factors related to IPD remain to be fully elucidated.
IPD as a disorder of iron metabolism. Of the total iron in an adult, 25% is a "reserve" stored in the cytoplasm as ferritin. The remaining iron is transported into mitochondria for synthesis of mitochondrial electron transport proteins or heme for production of cytochromes, hemoglobin or myoglobin (18). In IPD, iron accumulates in the substantia nigra (19-21) and receptors for the iron-transport protein lactoferrin are increased (22), while ferritin concentrations are reduced (23-25). This imbalance of "free" iron is believed to accelerate free radical formation and lipid peroxidation. It was recently found that lower concentrations of serum total iron binding capacity (TIBC), transferrin, iron and ferritin are detected in patients with IPD compared to controls. A paradoxical relationship between these parameters indicates a subtle, yet systemic, perturbation of iron metabolism. Low ferritin and serum iron suggested low iron stores, while the reduction in TIBC and transferrin, implied increased iron deposition.
These incongruous observations, at first, implied that the iron-regulatory proteins (IRP) which regulate the synthesis of iron-related proteins at the translation level (27) do not function normally in IPD, leading to increased iron entry into neurons, lower levels of iron in the extracellular compartment and a redistribution of free iron to the nigral intracellular compartment.
However, while attempting to identify the mechanism of this alteration in iron homeostasis, we discovered, by non-denaturing polyacrylamide gel electrophoresis of IPD and control sera, a shifted band which may comprise a variant of a nuclear-encoded mitochondrial gene, mitochondrial aconitase hydroxylase (ACO2). ACO2 is an iron-sulfur enzyme of the citric acid cycle responsible for the interconversion of citrate and isocitrate (FIG. 1). Because ACO2 is known to have relatively rare isoforms (28), we now infer that this protein, whose shift we have confirmed by Western blot using antibody, to bovine ACO2, may impart susceptibility to IPD via either mutation in the gene itself, mRNA splice variation, post-translational modification or non-covalent modification (e.g. a conformational change). Whatever it's molecular nature, the shifted band appears in 28/30 (93%) of IPD patients and in none of the controls. We have also recently discovered that the major isoform of human ACO2 in brain differs from that in placenta, suggesting brain-specific isoforms. Surprisingly, the genetic locus for ACO2 is on chromosome 22q13 near the CYP2D6 region (29). In parallel work, we excluded CYP2D6 as a candidate gene for IPD, but we did find evidence for an allelic association between IPD and dinucleotide repeat markers in the region near CYP2D6. That region includes the ACO2 gene. We now plan to sequence ACO2 in patients and controls to identify a potential mutation in IPD. We also recognize that a variant of ACO2 may be the result of a post-translational modification and not the result of a mutation or splice variant, or could be a consequence of an alteration induced by nitric oxide damage (31). As will be clear in our methods, we will not rely on any singular method of investigation to outline the putative role of ACO2 in the pathogenesis of IPD.
IPD as a disorder of mitochondria. Activity in complex I of the respiratory chain is reduced in the substantia nigra of patients with IPD (32, 33), particularly for subunits encoded in the mitochondrial genome (34, 35). MPTP inhibits the NADH-coenzyme Q reductase (complex I) activity in mitochondria (36-38). Swerdlow et al (39) have demonstrated that mitochondria from patients with IPD transfected into a cell line devoid of mitochondria gradually lose complex I activity. Mitochondrial DNA (mtDNA) is compact and encodes 13 enzymes of the respiratory chain complex (40). Mitochondria consume O.sub.2 and in the process generate free radicals which react with membrane lipids, nucleic acids and proteins. The cause of neuronal death in IPD is unknown, but it has been proposed that oxidative damage to mtDNA may play a role (41). However, mtDNA mutations accumulate with age (42) and the consequent deficit in ATP production in certain critical neuronal populations could also indirectly contribute to the pathogenesis of IPD. Mitochondrial energy deficits have been postulated to contribute to neuronal injury via excitotoxic mechanisms that include oxygen free radical formation (43), glutamate (44) and nitric oxide toxicity (45). Even under normal circumstances, iron accumulates in the substantia nigra as a cofactor required for dopamine synthesis. However, excess iron accumulates in IPD (46); this likely shifts iron into mitochondria, leading to oxidation of mitochondrial nucleic acids.
Evidence that IPD is associated with higher energy expenditure is supported by recent findings (47) and has been confirmed by others (48). IPD patients consume 25% more calories than controls, suggesting a systemic alteration in energy metabolism. Yet during the course of the illness IPD patients typically lose weight while becoming less physically active. It is possible that the dietary alterations could have a role in the pathogenesis of IPD by contributing to lipid peroxidation (49, 50). Alternatively, changes in diet may actually be a response to the higher metabolic rate induced by disease.
If ACO2 plays a central role in the pathogenesis of IPD, as we believe, how can we explain its influence on both energy and iron metabolism in IPD? Cytosolic aconitase (ACO1) controls iron homeostasis by modulation of the translation of an array of iron proteins including transferrin, transferrin receptor, ferritin, d-ALA-synthetase and even ACO2, all of which are encoded in the nuclear genome (27, 51, 52). Synthesis of these proteins is controlled by cytoplasmic iron status. The mRNAs of each of these proteins contain a non-translated loop of nucleotides referred to as an iron-binding element or iron-response element (IRE). The consensus stem of the IRE contains 21 nucleotides, only one of which is highly conserved. The apex of the loop contains six nucleotides; the first five are highly conserved (CAGUG) and the sixth is usually a pyrimidine (53, 54). Translation of ferritin, transferrin receptor and ACO2 mRNA is largely controlled by an iron-binding cytosolic protein (known as IRE-BP, IRP1 or ACO1) which binds to the IRE within these mRNAs when cytoplasmic iron is insufficient. Amino acid sequencing of IRP1 (ACO1) revealed 57% homology with ACO2 (55). Both ACO1 and ACO2 contain a 4Fe-4S cubane cluster which serves as the enzymatic binding site for citrate (FIG. 2, Part A). When iron is insufficient, ACO1 assumes a 3Fe-4S configuration, loses its enzymatic activity and is transformed into an mRNA-binding-protein which then attaches to the mRNA IRE (FIG. 2, Part B). For example, binding of ACO1 to the transferrin receptor mRNA IRE increases the synthesis of the transferrin receptor protein; binding to the mRNA IRE of ferritin stops its synthesis. Thus, ACO1 is able to respond to iron deficiency and regulate iron homeostasis. We postulate that ACO2 plays a similar role by regulating the synthesis of proteins encoded by the mitochondrial genome. While the gene for ACO2 itself is in the nuclear genome, many proteins involved in mitochondrial energy metabolism, e.g. those of complex I, reside in the mitochondrial genome. We have already searched the mitochondrial genome for evidence of an IRE comparable to those of ferritin and transferrin receptor, as described above, but none exists. We propose that the mRNAs of proteins encoded in the mitochondrial genome may have a different IRE structure for specifically binding ACO2. In this manner, ACO2 may regulate the synthesis of proteins of complex I and may, in turn, regulate mitochondrial iron uptake from the cytoplasm.
Strong indirect evidence in support of our ACO2 hypothesis is derived from the bacteriology literature. Microbial iron chelators have been studied for years as candidate drugs for the treatment of hemosiderosis. When iron is deficient, bacteria use citrate to synthesize highly-specific iron chelators; they simultaneously synthesize membrane receptors which recognize the iron chelator complex (56). Parenthetically, we note that citrate itself is an important intracellular iron chelator in all cells. During development, bacteria express at least two isoforms of aconitase; each isoform is associated with a different rate of iron uptake by the organism (57). Moreover, man-made bacterial mutants which cannot grow on iron deficient medium (i.e. cannot make iron chelators from citrate, see 58, 59), cannot grow at all on citrate and thus appear to be aconitase mutants; they are also resistant to manganese (Mn) toxicity (56). In short, both citrate and aconitase play critical roles in microbial iron and energy metabolism. In light of our evidence for an altered form of ACO2 in IPD, and the location of the ACO2 gene on chromosome 22q13, we feel that the ACO2 "isoform" hypothesis deserves considerable attention.
Finally, manganese (Mn), which induces levodopa-responsive parkinsonism (9, 10), is a potent inhibitor of ACO2. Previous investigators have demonstrated that Mn can occupy the fourth Fe site (i.e. the citrate binding site) in the enzyme active site (FIG. 2, Part C) (60). Using commerically available purified pig aconitase, we have recently observed that Mn alters aconitase Km but not Vmax. Thus, one may reasonably conduct experiments to determine whether patients with IPD have altered "endogenous" ACO2 kinetics; such altered kinetics could contribute to altered mitochondrial energy and iron metabolism.
It is unclear how iron is regulated in mitochondria, but both ATP and citrate appear to play a role (61). We propose that ACO2 plays a role in mitochondria similar to the role ACO1 plays in the cytosol. In our model, an altered ACO2 would be more often in the open configuration, where it loses aconitase activity and acts as an mRNA binding protein (FIG. 2, Part B). ACO2 would bind to mitochondrial DNA encoded mRNAs, thus altering protein synthesis. As a result, decreased levels of Complex I would occur, which is a known manifestation of IPD (32-35). At the same time, progression through the citric acid cycle would be decreased due to slightly reduced aconitase activity (FIG. 1), altering cellular metabolism. The altered form of ACO2 would lead to increased synthesis of a not-yet-identified mitochondrial membrane equivalent of the cell membrane transferrin receptor; such receptor would increase iron uptake into mitochondria, ultimately leading to free radical generation and cell death. In support of this hypothesis are these four points: 1) ACO2 has a 4Fe-4S cubane cluster; 2) there appears to be a defect in mitochondria in IPD; 3) there are increased iron levels in substantia nigra in IPD; and 4) there may be isoforms of ACO2 specific to brain (see below) with potentially different activities.